Chromogenic dibenzoxazepinone and dibenzothiazepinone enzyme substrates

ABSTRACT

Chromogenic enzyme substrate compounds comprising a dibenz[b,e] [1,4]oxazepinone or dibenzo[b,e] [1,4]thiazepinone nucleus having an enzyme-cleavable group such as a radical of a sugar, carboxylic acid, amino acid, peptide, phosphoric acid, or sulfuric acid. The substrate compounds are, in general, highly soluble in aqueous media and only slightly colored, and produce, upon enzyme cleavage, a chromogen exhibiting a large change in absorbance and a pKa below 7. Such substrates find use as indicators for the determination of enzyme analytes and enzymes used as markers in a variety of assays, including immunoassays.

This is a division of application Ser. No. 364,157, filed on Jun. 12,1989, now allowed.

BACKGROUND OF THE INVENTION

The present invention relates to chromogenic compounds which are usefulas optical indicator compounds in analytical test systems. Inparticular, the present invention relates to novel chromogenic enzymesubstrate compounds and their use in analytical test systems for thedetection of enzymes in a liquid test sample.

The determination of enzymes is important in a variety of fields such asbiochemical research, environmental and industrial testing, and medicaldiagnostics. The quantitation of enzyme levels in body fluids such asserum and plasma provides very useful information to the physician indiagnosing diseased states and their treatment. In addition to beinganalytes of interest in biological fluids, enzymes can also serve asdetection reagents in a variety of analytical systems such asimmunoassays and nucleic acid hybridization techniques. In such systems,enzymes are useful directly or indirectly as labels to monitor theextent of antigen-antibody binding or nucleic acid hybridization thatoccurs.

Accordingly, the desire to detect enzyme analytes and to use enzymelabels as a diagnostic tool in various analytical test systems has givenrise to the development of optical indicator compounds for use in thedetection and measurement of the activity of such enzymes. Typically,such known optical indicator compounds comprise a detectable chemicalgroup, such as a fluorogen or a chromogen, which has been derivatizedwith an enzyme cleavable substrate group specific for the enzyme ofinterest. Such optical indicator compounds exhibit an optical signalwhich is different from the optical signal which is provided by thecleaved native form of the fluorogen or chromogen. In principle, theenzyme cleaves the indicator compound to liberate the fluorogen orchromogen in the form of a distinctly fluorescent or colored product toprovide a change in fluorescence or color which is proportional to theamount of enzyme present which, in turn, can be correlated to the amountof analyte present in a liquid test sample.

In particular, the detection and/or determination of hydrolases, i.e.,enzymes which catalyse hydrolysis reactions of esters, glycosidic bonds,peptide bonds, other carbon-nitrogen bonds, and acid anhydrides [seeLehninger, Biochemistry (Worth Publishers, Inc., New York, N.Y., 1970)p. 148], is of interest in the diagnosis and monitoring of variousdiseases such as, for example, the determination of amylase and lipasein the diagnosis of pancreatic dysfunction [see Kaplan and Pesce,Clinical Chemistry--Theory, Analysis and Correlation (C. V. Mosby Co.,St. Louis, Mo., 1984) Chapter 56], determination ofN-acetylglucosaminidase (NAG) as an indicator of renal disease [seePrice, Curr. Probl. Clin. Biochem. 9, 150 (1979)], and detection ofesterase as an indicator for leukocytes [see Skjold, Clin. Chem. 31, 993(1985)]. Further to their value in disease monitoring, hydrolases inrecent years have gained importance in the diagnostic as well as in thebiotechnology areas. For example alkaline phosphatase and, preferably,β-D-galactosidase have found increasing use as indicator enzymes forenzyme immunoassays [see Annals of Clinical Biochemistry 16, 221-40(1979)].

Accordingly, the use of enzymes such as glycosidases, particularlyβ-D-galactosidase, as indicator enzyme labels in analytical test systemshas given rise to the development of substrate glycosides such asphenyl-β-D-galactoside, o-nitrophenyl-β-D-galactoside andp-nitrophenyl-β-D-galactoside [see Biochem. Z., Vol. 333, p. 209 [1960)]which are hydrolysed by β-D-galactosidase to liberate the phenols whichare determined photometrically in the ultraviolet range, or thenitrophenols which are determined in the shortwave visible range,respectively. European Patent Publication No. 156,347 and U.S. Pat. No.4,810,636 describe glycosides of resorufin and acridinone derivatives,respectively, which are specific for and cleaved by the particularglycosidase of interest to liberate detectable chromogens. U.S. Pat. No.3,950,322 describes an N-acyl-neuraminic acid derivatized with afluorogen such as 4-methylumbelliferone, fluorescein, methylfluorescein,resorufin, or umbelliferone for the detection of neuraminidase where thefluorogenic substrate glycoside is similarly acted upon by the enzyme toliberate the fluorogen.

The use of β-D-galactosides has also been described in conjunction withhistochemical investigations, such as the napthyl-β-D-galactosidesdescribed in Histochemie, Vol. 35, p. 199 and Vol. 37, p. 89 (1973), andthe 6-bromo-α-napthyl derivatives thereof described in J. Biol. Chem.,Vol. 195, p. 239 (1952). According to such test systems, the naptholswhich are liberated upon the interaction of the galactoside with theenzyme are reacted with various diazonium salts to yield the respectiveazo-dyes which can then be visualized.

Although such known optical indicator compounds are useful for thedetection of enzyme analytes and labels in an analytical test system, anumber of problems nevertheless exist which effect assay sensitivity andaccuracy such as low extinction coefficients, poor water solubility,absorbance maxima which interfere with various pigments and otherconstituents commonly present in biological fluids, and color shiftsbetween the optical indicator compound and the liberated chromogen orfluorogen which are difficult to measure without the use of complicatedinstruments.

Accordingly, it is an object of the present invention to providechromogenic enzyme substrate compounds which can be employed as opticalindicator compounds in analytical test systems for the accurate andsensitive determination of enzymes in a liquid test sample.

Further, it is an object of the present invention to provide chromogenicenzyme substrate compounds which can be incorporated into the solid,porous matrix of an analytical test device as optical indicatorcompounds for the measurement of enzymes incorporated therein or in aliquid test sample applied thereto.

SUMMARY OF THE INVENTION

The present invention provides novel chromogenic enzyme substratecompounds of the formula: ##STR1## where Y represents anenzyme-cleavable group which is selected to confer specificity to aspecific corresponding enzyme of analytical interest; W is oxygen orsulfur; and R and R', which can be the same or different, are hydrogen,alkyl, or aryl. The enzyme-cleavable group Y is a radical of a compoundY-OH comprising an enzyme-specific moiety which can be selected toconfer specificity to any one of a wide variety of enzymes and includes,but is not necessarily limited to, enzyme-specific moieties such assugars and derivatives thereof, acyl groups including aliphatic andaromatic carboxylic acids, amino acids and peptides, and inorganic acidssuch as phosphoric and sulfuric acids.

The present invention derives its principal advantages from the use ofdibenzoxazepinone and dibenzothiazepinone chromogens as intermediateswhich are derivatized with an appropriate enzymatically-cleavable groupY. In particular, when the enzymatically-cleavable group Y is cleaved bya specific enzyme therefor in a basic solution, preferably from betweenabout pH 7.0 to pH 10.0, a deprotonated form of the chromogen isliberated having an absorbance maximum which is substantially greaterthan the absorbance maximum of the chromogenic enzyme substrate compoundof the present invention whereby a distinct change in absorbancetherebetween is provided. The distinct change in absorbance provides areadily observable and detectable optical signal which can be accuratelymeasured and correlated to the amount of enzyme present in a liquid testsample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram of the synthetic pathway for the preparation of8-hydroxy-11H-dibenz[b,e][1,4]oxazepin-2-one chromogens.

FIG. 2 is a flow diagram of the synthetic pathway for the preparation of8-hydroxy-11H-dibenzo[b,e][1,4]thiazepin-2-one chromogens.

FIG. 3 is a flow diagram of the synthetic pathways for the preparationof chromogenic enzyme substrate compounds of the present invention.

FIG. 4 is a graph which illustrates the dose response of a test deviceincorporated with the chromogenic enzyme substrate of the presentinvention to the presence of β-D-galactosidase.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The chromogenic enzyme substrate compounds of the present invention arederived from chromogens having the general formula: ##STR2## Where W isoxygen, the chromogen will be a mixture of the isomers8-hydroxy-11H-dibenz[b,e][1,4]-oxazepine-2-one and2-hydroxy-11H-dibenz[b,e][1,4]-oxazepine-8-one (such 0-analog chromogensand their derivatives will be referred to herein as dibenzoxazepinones).Where W is sulfur, the chromogen will be a mixture of the isomers8-hydroxy-11H-dibenzo[b,e][1,4]thiazepin-2-one and2-hydroxy-11H-dibenzo[b,e][1,4]thiazepin-8-one (such S-analog chromogensand their derivatives will be referred to herein asdibenzothiazepinones). The dibenzazepinone where R is H and R' is methylhas been described in the literature [R. Hill, Journal of Bioenergetics,vol. 4, p. 229 (1973) and R. Hill, et al., New Phytology, vol. 77, p. 1(1976)]. The visible absorption spectra of this chromogen has beendescribed by T. Graan, et al., Analytical Biochemistry, vol. 144, p. 193(1985), where a 122 nm shift in absorption (λ_(max)) between theprotonated form and the deprotonated form of such chromogen wasreported. Such deprotonation occurs in weakly acidic solutions, usuallyfrom between about pH 5.75 to pH 6.75, at the phenolic hydroxyl group ofthe chromogen by delocalization of the negative charge of the anionthroughout the molecule. In the case of the present enzyme substratecompounds (1), enzymatic cleavage of the Y residue followed bydeprotonization produces the chromogenic species: ##STR3## where W, R,and R' are as defined above.

According to the teachings of the present invention, when the phenolichydroxyl group of the chromogen is derivatized with anenzymatically-cleavable group comprising a radical of a compound Y--OHwhich is an enzyme-specific moiety, the resulting compounds are novelisomeric chromogenic enzyme substrate compounds of the general isomericformula: ##STR4## wherein Y represents the enzyme-cleavable group, andW, R, and R' are as defined above (hereinafter, references to compoundsof the present invention by the use of only one of the two isomericstructures depicted in any of formulas (1) through (4) shall beunderstood to include reference to the other isomeric structure aswell). The isomeric forms of the present substrate compounds can be usedas a mixture, or can be separated by conventional means such aschromatography. The dibenzoxazepinones where W is O are particularlypreferred. Moreover, it is preferred that R and R' be selected from H,lower alkyl, and phenyl, including substituted forms thereof. When oneof R and R' is H or phenyl, it will generally be preferred that theother not also be H or phenyl, respectively. Particularly preferred arethe dibenzoxazepinones (W═O) where R and R', same or different, are H orlower alkyl, especially where one of R and R' is H and the other islower alkyl, e.g., methyl, or where both R and R' are methyl.

It should be understood that the present invention describes the firstuse of the dibenzoxazepinone and dibenzothiazepinone classes ofchromogens as indicator groups in chromogenic enzyme substrates and,accordingly, encompass a wide variety of substituted dibenzoxazepinoneand dibenzothiazepinone derivatives. It will be evident that thearomatic rings A and B in the formula (4) can bear a variety ofsubstituent groups without departing from the scope of the presentinvention. As discussed in greater detail hereinafter, such substituentgroups are limited only by the ability of one of ordinary skill in theart to prepare stable compounds which have the chromogenic enzymesubstrate properties of the present invention, and include such groupsas unsubstituted and substituted alkyl, unsubstituted and substitutedaryl, alkoxy, aryloxy, halo (e.g., fluoro, chloro, bromo), nitro andsubstituted amino such as dialkylamino.

In the context of the present invention, "alkyl" is intended to includelinear and branched forms of unsubstituted hydrocarbon residues of thegeneral formula--C_(n) H_(2n+1), preferably of the "lower alkyl"aliphatic type wherein n is 6 or less, such as methyl, ethyl, n-propyl,iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl, and the like, aswell as substituted forms thereof.

Further, in the context of the present invention "aryl" is intended toinclude organic residues derived from an aromatic hydrocarbon ring orring system by removal of a hydrogen atom, and include the unsubstitutedhydrocarbon ring residues such as phenyl and napthyl, and substitutedforms thereof. For purposes of the present invention, aryl residuesinclude those bearing one or more same or different functional groups orsubstituents which can be selected by one skilled in the art to providethe chromogenic enzyme substrate compounds of the present invention.

More particularly, where "aryl" and "alkyl" are substituted, suchsubstitution is intended to include such groups or substituents whenmono- or polysubstituted with functional groups which do notsubstantially detract from the useful features of the present compounds.Such functional groups include chemical groups which may be introducedsynthetically and result in the stable and useful chromogenic enzymesubstrate indicator compounds of the present invention. Examples of suchfunctional groups include, but are not intended to be limited to, halo(e.g., fluoro, chloro, bromo), substituted amino such as dialkylamino,nitro, alkoxy, aryloxy, alkyl, and aryl.

In particular, where R and/or R' are alkyl, preferably lower alkyl, suchalkyl groups include, but are not intended to be limited to, methyl,ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl,and substituted forms thereof including, but not necessarily limited to,benzyl, dialkylaminomethyl, more particularly dimethylaminomethyl, orhalomethyl, more particularly bromomethyl, and the like. Where R and/orR' are aryl, such aryl groups include, but are not intended to belimited to, napthyl, phenyl, p-chlorophenyl, 2,4-dimethoxyphenyl, andthe like.

The chromogenic enzyme substrate compounds (1) possess essentially thesame color properties as the protonated form of the chromogen,regardless of the pH of the surrounding liquid environment, wherein uponcontact of the derivatized chromogenic enzyme substrate compound (1)with an appropriate enzyme in a surrounding environment comprising asolution from between about pH 6.5 to pH 10, the enzymatically-cleavable group Y is cleaved by the enzyme to liberate the dissociatedor deprotonated form of the chromogen (3) having an absorbance maximumwhich is substantially greater than the absorbance maximum of thechromogenic enzyme substrate compound to provide a distinct change inthe absorbance maximum therebetween. Accordingly, the chromogenic enzymesubstrate compounds of the present invention are particularly useful inan analytical test system which requires the detection of anenzyme-labeled assay reagent employed therein. The distinct andmeasurable change in the absorbance maximum which is generated betweenthe substrate compound and the deprotonated form of the chromogen can beaccurately detected, measured and correlated to the amount of analytepresent in a liquid test sample.

Enzymatically-Cleavable Groups

According to the present invention, the enzyme-cleavable group Y is aradical of a compound Y--OH comprising an enzyme-specific moiety toprovide novel chromogenic enzyme substrate compounds which conferspecificity to a wide variety of enzymes encountered in analyticalchemistry, particularly clinical chemistry, and particularly hydrolases.The compound Y--OH is intended to include, but is not necessarilylimited to, sugars and derivatives thereof, acyl groups includingaliphatic and aromatic carboxylic acids including amino acids andpeptides, and inorganic acids such as phosphoric and sulfuric acidgroups.

It is to be understood that it will be evident to one skilled in the artthat the selection of the enzymatically-cleavable group Y will depend,of course, upon the particular enzyme of interest. For example, wherethe enzyme of interest is a glycosidase, a glycoside can be prepared inwhich the enzymatically-cleavable group Y is the glycosidic radicalcorresponding to the natural substrate for the particular glycosidase.Suitable glycosidic radicals include, but are not intended to be limitedto, mono- and oligosaccharide radicals, which are capable of beingincorporated into a glycoside substrate specific for a particularglycosidase enzyme and cleaved by said enzyme, such as radicals ofβ-D-galactopyranose, α-D-galactopyranose, β-D-glucopyranose,α-D-glucopyranose and α-D-mannopyranose, as well as amino sugars such asN-acetylglucosamine and N-acetylneuraminic acid, and the like radicals.Other suitable glycosidic radicals include oligosaccharide chains frombetween about 2 to 20, preferably 2 to 7, monosaccharide units attachedby α-1-4 glucosidic linkages, which can be broken down bysaccharide-chain splitting enzymes to a mono- or oligosaccharide which,in turn, can be cleaved by a corresponding glycosidase, such as, forexample, radicals of maltopentose, maltohexose and maltoheptose.

It is to be understood that in some instances where the glycosidicradical is an oligosaccharide chain as heretofore described, such chainis first modified or broken down to a shorter oligosaccharide ormonosaccharide by the enzyme under determination to produce a secondarysubstrate compound in which the enzymatically-cleavable group is cleavedfrom the nucleus of the substrate compound by a secondary enzyme, inwhich case the secondary substrate compound is then contacted with thesecondary enzyme to generate a measurable change in absorbance asheretofore described. For example, where the enzyme under determinationis α-amylase, the oligosaccharide chain is cleaved to produce asecondary glycoside substrate compound, e.g., an α-glucoside orβ-glucoside, in which the resulting glycoside group thereof is cleavablefrom the nucleus of the substrate compound by a secondary glycosidaseenzyme, e.g., α-glucosidase or β-glucosidase, respectively.

In the case of nonspecific esterase enzymes, the enzymatically-cleavablegroup Y is a radical of an acyl group to provide a chromogenic ester ofthe formula: ##STR5## Where Z is lower alkyl or aryl, such compounds canbe employed for the detection of nonspecific esterase enzymes such ascholinesterase, acylase, lipase, and the like. The chromogenic enzymesubstrate compounds of the present invention can also be utilized forthe detection of proteolytic enzymes commonly found in leukocytes. Insuch compounds is a radical of the compound Y--OH which is anN-protected amino acid or short peptide, e.g., consisting of betweenabout 2 to 5 amino acid units. For example, Y can be a radical of theN-protected amino acid N-tosyl-L-alanine as represented by the formula:##STR6## It will be appreciated that the present invention contemplatesother carboxylic acid residues, amino acid residues and N-protectinggroups as equivalents, as will be described in greater detail hereafter.

Similarly, for the detection of alkaline phosphatase from a liquid testsample, the enzymatically-cleavable group Y is a radical of the compoundY--OH wherein Y--OH is a phosphoric acid group of the formula: ##STR7##

Preparation of Chromogenic Enzyme Substrate Compounds

The chromogenic enzyme substrate compounds (1) of the present inventioncan be prepared by reacting the compound Y--OH, where Y is a selectedenzymatically cleavable group, with an appropriately derivatizeddibenzoxazepinone or dibenzothiazepinone chromogen, as will be describedin greater detail hereinafter, under condensation reaction conditionsknown in the art. Generally, the appropriate dibenzoxapeinone ordibenzothiazepinone chromogen is coupled under appropriate conditionswith a reactive derivative of the compound Y--OH, preferably acarbohydrate (sugar) or carbohydrate-derivative or an acid as heretoforedescribed, to provide a chromogenic enzyme substrate having the desiredstereoisomerism.

As stated above, the present invention contemplates various substituentswhich can be substituted at the aromatic rings A and B of the nucleusshown in formula (4). Substituted equivalents are prepared through theuse of appropriately derivatized dibenzoxazepinones ordibenzothiazepinones which can be prepared according to methods known inthe art.

The preparation of dibenzoxazepinones (FIG. 1) employs, as startingmaterials, a 3-hydroxyacetophenone, a 3-hydroxybenzophenone or a3-hydroxybenzaldehyde (8) and an appropriate Grignard reagent which arereacted [reaction (α)] to result in a substituted phenol (9).Alternately, the 3-hydroxyactophenone, 3-hydroxybenzophenone or3-hydroxybenzaldehyde (8) may be reduced [reaction (a')] using anappropriate reducing agent. The phenol (9), in turn, is reacted[reaction (b)] with a substituted benzoquinone-N-chloroimine (10) toresult in a functionalized indophenol (12). The indophenol (12) is thenallowed to cyclize in base to result in the desired substrate compound(13 and 14).

In particular, the phenols (9) are prepared according to the methoddescribed by Hill, et al., supra, where R and R' can both be methyl orphenyl and A, B and C are hydrogen, from the corresponding3-hydroxyacetophenone or 3-hydroxybenzophenone (8) which are reactedwith [reaction (α)] a methylmagnesium bromide Grignard reagent orphenylmagnesium iodide Grignard reagent respectively. It is to beappreciated that the Grignard reagent can be selected from a widevariety of such reagents which have been described in the art andinclude, but are not necessarily limited to alkyl and aryl Grignardreagents, such as where X represents bromine or iodine, as well as thosebearing functional group substituents such as -O-alkyl (alkoxy), -O-aryl(aryloxy), -alkyl and -aryl. Similarly, the synthesis of a variety ofsubstituted 3-hydroxyacetophenones (8) where R can be alkyl orsubstituted alkyl, and 3-hydroxybenzophenones (8) where R can be aryl orsubstituted aryl, have been described and include compounds of thegeneral formula (8) where R, A, B and C can be selected from a widevariety of substituents known in the art. For example, R can be methyl,A and C can be hydrogen, and B can be bromo, chloro, iodo, methyl orcyclohexyl [J. Med. Chem., Vol. 23, p. 738(1980)]; or R and C can bemethyl, A and B can be nitro and hydrogen or hydrogen and nitro,respectively, or A and B can be hydrogen [Chem. Ber., Vol. 92, p.2172(1959)]; or R can be methyl, A and C can be hydrogen, and B can bemethoxy [Chem. Ber., Vol. 55B, p. 1892(1922)] or cyclohexylether [J.Chem. Soc., p. 3430(1951)]; or R and A can be methyl, B can be hydrogen,and C can be nitro [J. Org. Chem., Vol. 14, p. 397(1949)]; or R can bemethyl, A and B can be methoxy, and C can be hydrogen [J. Prakt. Chem.,Vol. 103, p. 329(1922)]; or R can be methyl, A and C can be hydrogen,and B can be p-hydroxyphenol [Hoppe-Seyler's Z. Physiol. Chem., Vol.292, p. 58(1953)]; or A, B and C can be hydrogen, and R can bedimethylaminomethyl [Monatsh., Vol. 80, p. 517(1949)]or benzyl orphenylethyl [Medd. Norsk. Farm. Selskap., Vol. 24, p. 45(1962) orp-chlorophenyl [J. Chem. Soc., p. 5(1946)] or 2,4-dimethoxyphenyl [Bull.Soc., Chim. France, p. 1682(1959)]; or R can be bromomethyl and where A,B or C is nitro, then B and C, A and C or A and B can be hydrogen,respectively [Acta Univ. Szeged., Acta Phys. Chem., Vol. 9, p.48(1963)]; or R can be phenyl and A and C can be hydrogen and B can bemethyl [Helv. Chim. Acta., Vol. 29, p. 1413(1946)]or A and B can bemethoxy and C can be hydrogen [J. Org. Chem., Vol. 24, p. 952(1959)];and the like.

Phenols (9) in which either or both R and R' are H are prepared from thecorresponding 3-hydroxybenzaldehyde, 3-hydroxyacetophenone or3-hydroxybenzophenone (8) by reduction [reaction (α')] of the carbonylgroup to a hydroxyl group using a variety of reducing agents. Suchreducing agents are known in the art (see House, Modern SyntheticReactions, 2nd edition, W. A. Benjamin, Inc., Menlo Park, Calif., 1972,pp. 1-227) and include lithium hydride, lithium aluminum hydride, sodiumborohydride and catalytic hydrogenation.

The desired indophenol (12) is prepared by reacting the appropriatelysubstituted phenol (9) resulting from reaction (α) or (α') with anappropriately substituted benzoquinone-N-chloroimine (10) in aqueousalkali [reaction (b)] as described by Hill, et al, supra, where all ofA-G can be hydrogen, and as described more generally by Gibbs, et al.,Supplement No. 69 to the Public Health Reports, Washington, D.C. (1928),where all of substituents D, E, F and G in the general structure (10)can all be hydrogen, or D can be methyl and E, F and G can be hydrogen,or D, E and G can be hydrogen and F can be methyl, or D and E can bechlorine or bromine and F and G can be hydrogen, respectively. Analternate synthetic pathway for the preparation of the indophenol (12)is also described by Corbett, J. Chem. Soc. (B), p. 1502 (1970) where anappropriately substituted phenol (9) is reacted with an appropriatelysubstituted p-aminophenol (11) and oxygen in the presence of aqueousalkali [reaction (o)]. The substituents D, E, F and G of thep-aminophenol (11) are described where D, E and G can be hydrogen and Fcan be methyl or chlorine, or D, F and G can be hydrogen and E can bemethyl, or D and G can be methyl and E and F can be hydrogen, or D and Ecan be methyl or chlorine and F and G can be hydrogen, or D can bechlorine and E, F and G can be hydrogen, respectively.

The indophenol (12) resulting from either reaction (b) or (c) is thenemployed to prepare the substrate compounds (13) and (14) according tothe method described by Hill, et al., New Phytology vol. 77, page 1(1976), [reaction (d)] where the indophenol (12) is cyclized (step 2) inaqueous base, preferably sodium borate (borax), for several days atambient temperature. It is to be appreciated that it is not necessary toisolate the various intermediates resulting from steps 1-3 of reaction(d) to obtain satisfactory yields of the substrate compounds (13) and(14).

The preparation of dibenzothiazepinones (FIG. 2) employs as startingmaterials substituted 3-mercaptomethylphenols (16) which are obtainedfrom the previously described substituted 3-hydroxymethylphenols (9) viaa two step process consisting of bromination [reaction (a)] to affordsubstituted 3-bromomethylphenols (15) followed by reaction with thioureaand base hydrolysis [reaction (a')]. The phenols (16) are in turnreacted [reaction (b)] with a substituted benzoquinone-N-chloroimine(10) to result in a functionalized indophenol (17). The indophenol (17)is then allowed to cyclize in base to result in the desired substratecompound (18 and 19).

In particular, the synthesis of 3-hydroxybenzyl bromide (15,R═R'═A═B═C═H) from 3-hydroxybenzyl alcohol (9, R═R'═A═B═C═H) isdescribed in J. Med. Chem. vol. 23, p. 1013 (1980), and its conversionto 3-mercaptomethylphenol (16, R═R'═A═B═C═H) is reported in J. Chem.Soc. Perkin I., p. 1555 (1980). More generally, transformation of avariety of substituted 3-hydroxy-benzyl alcohols (9) to thecorresponding 3-mercaptomethyl-phenols (16) with this procedure or withother appropriate procedures is within the ordinary skill in the art.

The specific nature of substituted benzoquinone-N-chloroimines (10) andsubstituted p-aminophenols (11) are the same as previously described,and the remaining steps (b), (c) and (d) are the same as thosepreviously described for the dibenzoxazepinones (13 and 14).

It is to be appreciated that selection of appropriately derivatizedstarting materials and an appropriate Grignard reagent or reducing agentresults in a variety of substituted phenols and, accordingly, oneskilled in the art of organic chemical synthesis can prepare specificindophenols having a variety of substituents which can be converted to adesired appropriately derivatized dibenzoxazepinones anddibenzthiazepinones for use as the chromogen of the chromogenicacridinone enzyme substrate compounds of the present invention.

The glycoside derivatives of the general formula (1) can be preparedaccording to methods known in the art of carbohydrate chemistryemploying known derivatives of carbohydrates of the formula Y--OH whichare reacted with an appropriate chromogen. Such carbohydratederivatives, which in some instances carry protecting groups, arecommercially available (Aldrich Chemical Co., Milwaukee, Wis., USA;Sigma Chemical Co., St. Louis, Mo., USA), or can be prepared accordingto methods known in the art [Methods in Carbohydrate Chemistry (AcademicPress, 1963), Vol. 2]. Glycosidic radicals include, but are not intendedto be limited to, radicals of sugars such as β-D-galactopyranose,α-D-galactopyranose, β-D-glucopyranose, α-D-glucopyranose,α-D-mannopyranose, N-acetylglucosamine, β-glucuronic acid and neuraminicacid. Other suitable glycosidic radicals include radicals ofoligosaccharide chains which by saccharide-chain splitting enzymes canbe broken down to the level of a mono- or oligosaccharide, which in itsturn can be directly split off from the nucleus of the substratecompound with the corresponding glycosidase. It is to be understood thatsuch oligosaccharide chains are chains consisting of from about 2 toabout 20, preferably 2 to 7 monosaccharide units, such as maltopentose,maltohexose or maltoheptose. The chromogens of the general formula (2)are reacted with a mono- or oligosaccharide or a 1-halogeno-derivativethereof, where all hydroxyl groups are substituted with a protectinggroup according to methods known in the art of carbohydrate chemistry,to give per-O-substituted glycosides, from which the glycosidederivatives of general formula (1) are obtained by splitting off theprotective groups according to methods known in the art.

The appropriate chromogens are reacted with the per-O-substituted1-halogenosaccharides, preferably in the presence of proton acceptorssuch as alkali hydroxides or alkali carbonates, in aqueous acetone or(under phase transfer conditions) in a water/chloroform or water/benzenemixture. This procedure can furthermore be carried out by firstconverting the chromogens with alkali hydroxide or alcoholate intoalkali salts or, using possibly substituted amines, into ammonium salts,and then reacting these with the per-O-substituted 1-halogenosaccharides in dipolar aprotic solvents such as acetone,dimethylsulfoxide, dichloromethane, tetrahydrofuran ordimethylformamide. Furthermore in the synthesis of per-O-substitutedglycosides and per-O-substituted 1-halogenosaccharides, it is effectiveto use additives in the form of single silver salts or mixtures ofsilver salts, such as silver oxide, silver carbonate, silver carbonateon Celite® (Johns-Manville Corp., Denver, Colo., USA), silver triflateor silver salicylate, and/or of single mercury salts or mixtures ofmercury salts, such as mercury bromide, mercury cyanide, mercury acetateor mercury oxide, and/or of single cadmium salts or mixtures of cadmiumsalts such as cadmium carbonate or cadmium oxide, possibly with the useof drying agents such as calcium chloride, a molecular seive orDrierite® (W. A. Hammond Drierite Co., Xenia, Ohio, USA), in solventssuch as methylene chloride, chloroform, benzene, toluene, ethyl acetate,quinoline, tetrahydrofuran or dioxane. In the synthesis of α-linkedglycosides, the chromogen is melted with a saccharide whose hydroxygroups are substituted with a protective group, preferably anacetyl-group, in the presence of a Lewis acid, such as zinc chloride[see Chem. Ber. 66, 378-383 (1933) and Methods in Carbohydrate Chemistry(Academic Press, 1967) Vol. 2, pp. 345-347]. The temperature of thereaction is preferably between 80° and 130° C., more preferably between110° and 130° C. The resulting per-O-substituted glycosides likewise arenew compounds. Removing the protecting groups from the per-O-substitutedglycosides to form glycosides is performed according to methods known inthe art of carbohydrate chemistry [see Advances in Carbohydrate Chem.12, 157 (1976)], such as with the protective acyl-groups with sodiummethylate, barium methylate or ammonia in methanol. Suitable as a"protecting group" commonly used in carbohydrate chemistry is especiallyan acetyl, benzoyl, benzyl or trimethylsilyl-radical.

Derivatives of the general formula (1) where Y is the radical of anoligosaccharide chain of from about 2 to about 20 monosaccharide unitsattached via α-1-4 glucosidic linkages can additionally be prepared fromα- and β-chromogen glucosides by an enzymatic process first described byFrench, et al., J. Am. Chem. Soc. 2387 (1954), and later by Wallenfels,et al., Carbohydrate Research 61, 359 (1978), involving the transfer ofthe glucoside to a pre-formed polysaccharide chain by the enzyme(1-4)-α-glucan-4-glucosyltransferase (also known as cyclomaltodextringlucanotransferase; EC 2.4.1.19).

Ester derivatives of the general formula (1) can be prepared by methodsknown in the art of organic chemistry by reacting an appropriatechromogen with known derivatives of carboxylic acids of the formulaY--OH, where Y═Z--C(O)-- and where Z is defined the same as R and R'above. Such known derivatives of carboxylic acids of the formula Y--OHinclude, but are not intended to be limited to, amino acid residues,preferably residues of naturally-occurring α-amino acids in their L- orD-form or also in their racemic form, the residues of glycine, alanine,valine, leucine, isoleucine, phenylalanine and tyrosine being preferred,the L-forms thereof being more preferred. Any free hydroxyl groupspossibly present may be acylated and preferably acetylated. The peptideresidues in this definition of Y--OH are to be understood to be, forexample, amino acids or peptides from between about 2 to about 5 aminoacid units such as di-, tri-, tetra-, and pentapeptides, di- andtripeptides being preferred, the amino acid components thereof being theabove-mentioned amino acids. It is also to be understood that the aminogroups of such amino acids or peptides may be protected with nitrogenprotecting groups known in the art of peptide chemistry [see T. W.Green, Protective Groups in Organic Synthesis (J. Wiley and Sons, NewYork, N.Y., 1981), pp. 218-287] including, for example, acyl,oxycarbonyl, thiocarbonyl, sulphonyl, especially p-toluenesulphonyl(Tosyl, Ts), sulphenyl, vinyl, cyclohexenyl, and carbamoyl, especiallyt-butyl-(BOC) and benzyl-(CBz) carbamoyl radicals. Such esters may alsobe similarly prepared by reacting an appropriate chromogen with acarboxylic acid, amino acid or peptide, Y--OH as defined above, or withan appropriate reactive derivative thereof, employing methods known inthe art of organic chemistry [see J. March, Advanced Organic Chemistry:Reactions, Mechanism and Structure (McGraw-Hill Book Co., New York,N.Y., 1968) pp. 319-323]. The reactive derivatives used can be, forexample, acid chlorides or bromides, or mixed anhydrides conventionallyused in peptide synthesis, such as those with ethyl chloroformate, oractive esters such as those of N-hydroxysuccinimide.

Similarly, inorganic esters of the general formula (1) can be preparedaccording to methods known in the art of organic synthesis. The knownderivatives of inorganic acids Y--OH, such as phosphoric acid orsulfuric acid are reacted with the chromogen employing methods known inthe art of organic chemistry, such as shown in Koller and Wolfbeis,Monatsh. 116, 65 (1985) for inorganic esters of certain coumarins.

Analytical Test Systems

The chromogenic enzyme substrate compounds of the present invention areuseful in analytical test systems which require the measurement of theamount of enzyme present therein, particularly those analytical testsystems employing enzyme-labeled assay reagents. Such analytical testsystems include, but are not intended to be limited to, enzymeimmunoassays known in the art as competitive, sandwich and immunometrictechniques where the amount of enzyme label in a particular fractionthereof can be measured and correlated to the amount of analyte underdetermination obtained from a liquid test sample.

The use of specific binding substances, such as antigens, haptens,antibodies, lectins, receptors, avidin, and other binding proteins, andpolynucleotides, labeled with an enzyme have been recently developed andapplied to the measurement of substances in biological fluids (see, forexample, Clin. Chem., Vol. 22, p. 1232 (1976); U.S. Reissue U.S. Pat.No. 31,006; and U.K. Patent No. 2,019,308). Generally, such measurementdepends upon the ability of a binding substance, e.g., an antibody or anantigen, to bind to a specific analyte wherein a labeled reagentcomprising such binding substance labeled with an enzyme is employed todetermine the extent of such binding. Typically, the extent of bindingis determined by measuring the amount of enzyme label present in thelabeled reagent which either has or has not participated in a bindingreaction with the analyte, wherein the amount of enzyme detected andmeasured can be correlated to the amount of analyte present in a liquidtest sample.

The chromogenic enzyme substrate compounds of the present invention areparticularly useful in analytical test systems as heretofore describedwhere an analytical test device comprising a carrier matrix incorporatedwith the chromogenic enzyme substrate compound of the present inventionis employed, the nature of the enzyme-specific moiety thereof depending,of course, upon the particular enzyme being detected.

The nature of the material of such carrier matrix can be of anysubstance capable of being incorporated with the chromogenic enzymesubstrate compound of the present invention, such as those utilized forreagent strips for solution analysis. For example, U.S. Pat. No.3,846,247 describes the use of felt, porous ceramic strips, and woven ormatted glass fibers. As substitutes for paper, U.S. Pat. No. 3,552,928describes the use of wood sticks, cloth, sponge material, andargilaceous substances. The use of synthetic resin fleeces and glassfiber felts in place of paper is suggested in British Pat. No.1,369,139, and British Pat. No. 1,349,623 teaches the use of alight-permeable meshwork of thin filaments as a cover for an underlyingpaper matrix. This reference also teaches impregnating the paper withpart of a reagent system and impregnating the meshwork with otherpotentially incompatible reagents. French Pat. No. 2,170,397 describesthe use of carrier matrices having greater than 50% polyamide fiberstherein. Another approach to carrier matrices is described in U.S. Pat.No. 4,046,513 wherein the concept of printing reagents onto a suitablecarrier matrix is employed. U.S. Pat. No. 4,046,514 describes theinterweaving or knitting of filaments bearing reagents in a reactantsystem. All such carrier matrix concepts can be employed in the presentinvention, as can others. Preferably, the carrier matrix comprises abibulous material, such as filter paper, whereby a solution of thechromogenic enzyme substrate compound of the present invention isemployed to impregnate the matrix. It can also comprise a system whichphysically entraps the assay reagents, such as polymeric microcapsules,which then rupture upon contact with the test sample. It can comprise asystem wherein the assay reagents are homogeneously combined with thecarrier matrix in a fluid or semi-fluid state, which later hardens orsets, thereby entrapping the assay reagents.

In a preferred embodiment, the carrier matrix is a bibulous material inthe form of a zone or layer incorporated with the chromogenic enzymesubstrate compound of the present invention which is employed where aparticular assay is performed in a liquid environment employing aninsoluble assay reagent known in the art to physically separate the freespecies of the labeled reagent from the bound species of the labeledreagent. According to such assay system, an aliquot of liquid containingthe free species is removed and applied to the carrier matrix whereinthe chromogenic enzyme substrate compound incorporated therein interactswith the enzyme label of the labeled reagent of the free species fromthe liquid test sample to provide a detectable signal which can bevisibly observed and/or measured with an appropriate instrument, such asa spectrophotometer.

Similarly, a test device comprising two or more carrier matrices in theform of, for example, an uppermost layer or zone and a lowermost layeror zone can be employed. The lowermost layer of such test device can beincorporated with the chromogenic enzyme substrate compound of thepresent invention wherein a liquid test sample containing analyte underdetermination is applied to the uppermost layer of the device. Theanalyte which diffuses therein participates in the necessary bindingreactions to generate a free and bound (i.e., immobilized) species ofthe enzyme labeled reagent therein as heretoforedescribed. Accordingly,the free species of the labeled reagent so generated is free to migrateinto the lowermost layer where the enzyme label of the free speciescleaves the enzymatically-cleavable group of the chromogenic enzymesubstrate compound of the present invention incorporated therein toprovide a measurable, detectable signal as heretofore described.

The present invention will now be illustrated, but is not intended to belimited, by the following examples. Italicised numbers in parenthesisrefer to the structural formulae as used in the figures and/or thespecification.

EXAMPLES8-(Tetra-O-acetyl-β-D-galactopyranosyloxy)-11-methyl-11H-dibenz[b,e][1,4]oxazepin-2-one(21) and2-(Tetra-O-acetyl-β-D-galacto™pyranosyloxy)-11-methyl-11H-dibenz[b,f][1,4]-oxazepin-8-one(22)

A mixture of 8-hydroxy-11-methyl-11H-dibenz[b,e]1,4]oxazepin-2-one("methyl purple") (20) (0.2 g; 0.83 mmol), prepared according to themethod of Hill, et al, supra, acetobromogalactose (Sigma Chemical Co.,St. Louis, Mo. USA) (0.685 g; 1.66 mmol) and silver (I) oxide (Ag₂ O)(Aldrich Chemical Co., Milwaukee, Wis. USA) (0.425 g; 1.66 mmol) wasstirred at ambient temperature in anhydrous quinoline (6.25 mL) andethyl acetate (EtOAc) (2 mL) for 16 hours in a stoppered flask protectedfrom light. The reaction mixture was diluted into EtOAc (approximately40 mL), filtered through Celite (Johns-Manville Corp., Denver, Colo.USA) and extracted with small portions of 1M HCl until the extracts wereacidic (pH=1). The combined aqueous extracts were washed with EtOAc (25mL), then the combined EtOAc layers were washed with brine (20 mL),dried with sodium sulfate (Na₂ SO₄), filtered and evaporated to drynessin vacuo to give a golden-yellow foam (0.8 g). The crude product waschromatographed on silica gel (100 g) using 7.5% (v:v) acetone inchloroform solvent and the two bright yellow product bands (Rf=0.28 and0.34 on silica gel plates developed with acetone:chloroform [1:9]) werecollected, combined, and freed of solvent to give a mixture of the titlecompounds as an orange foam (9.44 g; 92%).

IR (KBr) cm⁻¹ 2985, 1756, 1641, 1618, 1575, 1511, 1437, 1372, 1230,1075.

¹ H NMR (DMSO-d⁶)δ: 1.4-1.7 (m, 3H), 1.9-2.2 (m, 12 H), 4.0-4.2 (m, 2H), 4.45-4.55 (m, 1 H), 5.1-5.4 (m, 4 H), 5.6-5.76 (m, 1 H), 5.85-5.9(m, 1 H), 6.4-7.7 (m, 5 H).

¹³ C NMR (DMSO-d⁶) ppm. 187.93, 187.51, 169.99, 169.93, 169.59, 169.27,159.48, 158.56, 157.73, 151.67, 144.71, 142.38, 142.00, 140.69, 137.00,136.48, 134.34, 134.11, 130.51, 130.16, 125.75, 125.64, 116.69, 116.63,113.04, 112.95, 110.99, 107.38, 96.94, 76.72, 75.03, 70.76, 70.16,68.19, 67.34, 61.59, 20.52, 17.62, 17.23 (17 coincident bands).

Analysis: Calculated for C₂₈ H₂₉ NO₁₂ :

C, 58.84; H, 5.11; N, 2.45;

Found: C, 58.61; H, 5.31; N, 2.29.

8-β-D-Galactopyranosyloxy-11-methyl-11H-dibenz[b,e][1,4]oxazepin-1-one(23) and2-β-D-galactopyranosyloxy-11-methyl-11H-dibenz[b,e][-1,4]oxazepin-8-one(24)

A solution of (21) and (22) (0.41 g; 0.72 mmol) in HPLC grade methanol(25 mL) was treated at ambient temperature with sodium methoxide (31 mg)and allowed to stir for 1.5 hours. The reaction was quenched by additionof acetic acid (approximately 25 μL) then evaporated to dryness in vacuoto give an orange solid. The crude product was chromatographed on silicagel (100 g) using 15% (v:v) methanol in chloroform solvent and thebright orange product band (Rf=0.25 on silica gel plates developed withmethanol: chloroform [1.4]) was collected and freed of solvent in vacuoto give a mixture of the title compounds as a red-orange solid. Vacuumdrying for 2 hours at 64° C. gave the analytical sample (0.195 g; 67%).

IR (KBr) cm⁻¹ 3328, 2925, 2876, 1639, 1612, 1571, 1508, 1384, 1245,1219, 1085, 896, 880, 823, 791.

¹ H NMR (DMSO-d⁶)δ: 1.4-1.7 (m, 3 H), 3.3-3.75 (m, 7 H), 4.5-4.75 (m, 5H), 5.85-5.90 (m, 1 H), 6.4-7.65 (m, 5 H).

¹³ C NMR (DMSO-d⁶) ppm. 187.95, 187.51, 161.18, 159.34, 158.67, 152.07,151.61, 151.00, 144.76, 142.52, 142.11, 140.06, 136.96, 136.36, 134.28,133.85, 130.31, 129.95, 125.00, 116.93, 116.84, 113.08, 113.04, 110.91,107.36, 100.76, 100.59, 76.65, 75.94, 75.18, 73.34, 73.24, 70.21, 68.27,68.19, 60.52, 60.44, 17.68, 17.30 (1 coincident band).

Analysis: Calculated for C₂₀ H₂₁ NO₈ :

C, 59.55; H, 5.25; N, 3.47;

Found: C, 59.57; H, 5.48; N, 3.21.

When dissolved in 50 mM phosphate buffer at pH 7.4 containing 5 mMmagnesium chloride (MgCl₂) compound (23)+(24) (mixture) had λ_(max) of454 nm (.sub.ε =22,000) and 344 nm (.sub.ε =14,000). In the presence ofβ-galactosidase, the substrate was cleaved to (20) at a rate (K_(cat))of 1.32×10⁴ mol. min⁻¹ /mol. active site and exhibited a K_(m) of 0.075mM.

8-Hydroxy-11,11-dimethyl-11H-dibenz[b,e][1,4]oxazepin-2-one (27) and2-hydroxy-11,11-dimethyl-11H-dibenz[b,e][1,4]oxazepin-8-one (28).

A solution of 2-(3'-hydroxyphenyl)-1-propanol (25) (2.20 g; 14.45 mmol)(prepared as described by Bruce, et al, J. Chem. Soc. (C), 1627 [1966])and Na₂ B₄ O₂.10H₂ O (borax) (28 g; 73.42 mmol) in H₂ O (200 mL)maintained at ambient temperature was treated withbenzoquinonechloroimide (26) (2.0 g; 14.13 mmol) (prepared as describedby Gibbs, et al, Supplement No. 69 to The Public Health Reports,Washington, D.C. [1928]) and tetrahydrofuran (5 mL), then allowed tostir for 7 days. The reaction was then acidified with 1M HCl andextracted four times with EtOAc (125 mL). The combined EtOAc layers werewashed with brine (100 mL), dried (Na₂ SO₄), filtered and evaporated todryness in vacuo. The residue was chromatographed on silica gel (200 g)using acetone:chloroform (1:9) solvent; the red product band (R_(f)=0.33) was collected and freed of solvent in vacuo to give a mixture ofthe title compounds as a red powder (72 mg).

IR (KBr) cm⁻¹ 1634, 1614, 1556, 1311, 1213, 1180, 878.

¹ H NMR (DMSO-d⁶ /ambient temperature)δ: 1.52 (br.s, 6 H), 5.8-7.7(v.br.m, 6 H), 10.75 (br.s,1 H).

¹ H NMR (DMSO-d⁶ /100° C.)δ: 1.53 (s, 6 H), 2.98 (v.br.s, 1 H) (OH),6.13 (br.s, 1 H), 6.57 (br.d, J=9.3 Hz, 1 H), 6.66 (br.s, 1 H), 6.73(v.br.d., J=9.1 Hz, 1 H), 7.28 (d, J=9.3 Hz, 1 H), 7.44 (d, J=9.0 Hz, 1H).

EIMS, m/e (relative intensity) 255 (M⁺, base), 240 (14.8), 226 (44.3),212 (41.8), 210 (23.7), 198 (26.8), 184 (30.3).

Analysis: Calculated for C₁₅ H₁₃ NO₃.1/4 H₂ 0:

C, 69.35; H, 5.24; N, 5.39;

Found: C, 69.54; H, 5.26; N, 5.38.

8-(Tetra-O-acetyl-β-D-galactopyranosyloxy)-11,11-dimethyl-11H-dibenz[b,e][1,4]oxazepin-2-one(29) and2-(Tetra-O-acetyl-β-D-galactopyranosyloxy)-11,11-dimethyl-11H-dibenz[b,e][1,4]oxazepin-8-one(30).

A mixture of (27) and (28) (0.103 g; 0.4 mmol), acetobromogalactose(0.333 g; 2 eq) and silver (I) oxide (Ag₂ O) (0.188 g; 2 eq) was stirredat ambient temperature in anhydrous quinoline (7 mL) and EtOAc (2 mL)for 18 hours in a stoppered flask protected from light. The reactionmixture was diluted with EtOAc (approximately 40 mL), filtered throughCelite and extracted three times with 1.0M HCl (30 ml each). Thecombined aqueous extracts were washed with EtOAc (40 mL) then thecombined EtOAc layers were washed with brine (40 mL), dried (Na₂ SO₄),filtered and evaporated to dryness in vacuo. The crude product waschromatographed on silica gel (93 g) using acetone:chloroform (7:93)solvent and the two yellow product bands (R_(f) =0.38 and 0.43 on silicagel plates developed with acetone:chloroform [1:9]) were collected,combined, and freed of solvent in vacuo to give a mixture of the titlecompounds as an orange foam (0.216 g; 92%).

IR(KBr)cm⁻¹ 1750, 1640, 1615, 1573, 1368, 1260, 1070, 955, 898.

¹ H NMR (CDCl₃)δ: 1.45-1.70 (m, 6 H), 2.0-2.2 (m, 12 H), 4.08-4.26 (m, 3H), 5.10-5.20 (m, 2 H), 5.45-5.55 (m, 2 H), 6.00-7.70 (m, 6 H).

¹³ C NMR (CDCl₃) ppm. 188.82, 188.50, 170.25, 170.04, 169.94, 169.26,159.91, 158.00, 156.55, 151.67, 151.63, 150.56, 146.45, 145.38, 141.78,140.19, 137.11, 136.59, 135.11, 130.70, 129.46, 129.31, 126.22, 124.96,155.33, 115.28, 113.81, 112.76, 109.03, 98.54, 98.42, 80.14, 79.78,71.29, 70.57, 68.32, 66.73, 61.42, 26.44, 26.23, 20.56 (17 coincidentbands).

8-β-D-Galactopyranosyloxy-11,11-dimethyl-11H-dibenz[b,e][1,4]-oxazepin-2-one(31) and2-β-D-galactopyranosyloxy-11,11-dimethyl-11H-dibenz[b,f][1,4]oxazepin-8-one(32).

A solution of (29) and (30) (0.21 g; 0.358 mmol) in HPLC grade methanol(20 mL) was treated at ambient temperature with sodium methoxide (22 mg)and allowed to stir for several hours. The reaction was quenched byaddition of acetic acid (23 μL) then evaporated to dryness in vacuo. Thecrude product was chromatographed on silica gel (60 g) usingmethanol:chloroform (15:85) solvent and the orange product band (R_(f)=0.23 on silica gel plates developed with methanol:chloroform [1:4]) wascollected and freed of solvent in vacuo to give the title compound as ared-orange powder (0.12 g; 80%).

IR(KBr)cm⁻¹ 3416, 2926, 1634, 1612, 1567, 1503, 1385, 1292, 1227, 1074,889.

¹ H NMR (DMSO-d⁶)δ:1.45-1.65 (m, 6 H), 3.30-3.75 (m, 6 H), 4.5 (v.br.d,J=1.4 Hz, 1 H), 4.68 (br.q., J=6.7 Hz, 1 H), 4.91 (v.br.s, 1 H), 4.99(t, J=6.9 Hz, 1 H), 5.23 (v.br.s, 1 H), 5.9-7.7 (m, 6 H).

¹³ C NMR (DMSO-d⁶) ppm. 187.91, 187.76, 161.47, 159.68, 156.35, 150.61,150.48, 150.31, 146.25, 145.62, 141.99, 140.91, 138.65, 137.23, 136.20,134.09, 130.33, 129.19, 124.75, 116.15, 114.69, 113.32, 113.01, 108.65,100.58, 100.42, 80.72, 79.99, 76.00, 75.78, 73.30, 73.24, 70.19, 68.26,68.17, 60.52, 60.38, 26.01 (4 coincident bands).

Analysis: Calculated for C₂₁ H₂₃ NO₈.11/2 H₂ O:

C, 56,75; H, 5.90; N, 3.15.

Found: C, 56.61; H, 5.82; N, 3.14.

Test Device

A test device sensitive to the presence of β-galactosidase in a testsample was prepared. The device comprised a small rectangular piece offilter paper mounted at one end of an oblong strip of polystyrene film.The paper was impregnated with various ingredients, including (23) and(24), a buffer and inorganic salt. A 2 inch wide strip of Whatman 54filter paper was immersed in an aqueous solution containing thefollowing:

0.6M NaEpps Buffer (pH=8.4)

4.0 mM MgCl₂

The paper was then dried overnight in air. Next the paper was immersedin a DMF solution containing:

15 mM (23)+(24)

The paper was then dried in air at 50°-80° C. A yellow test paper wasobtained.

The piece of the dried, impregnated paper was cut into a rectanglemeasuring 0.1 inch×0.4 inch and mounted at one end of an axiallyoriented polystyrene strip measuring 0.1 inch×3.25 inch. Mounting thepaper to the strip was achieved using double-stick double-faced adhesive(3M Company).

Solutions of β-D-galactosidase in pH 6.4 potassium phosphate/citratebuffer were prepared at 0.025, 0.05, 0.075, 0.20, 0.125 and 0.15 IU/mL.Three analytical test devices were dipped into each test solution. Therespective analytical test devices were then mounted in a SERALYZER®Reflectance Photometer (Miles, Inc., Elkhart, Ind., USA) and thereflectance of light from the test device containing the liberatedchromogen (20) was measured at 590 nm after 70-90 seconds wherein thereflectance values thereof were plotted against the respective testsample solution concentrations to reveal a linear dose response asexemplified in FIG. 4.

The present invention has been particularly described and exemplifiedabove. Clearly, many other variations and modifications of the inventioncan be made without departing from the spirit and scope thereof.

What is claimed is:
 1. A method for determining a particular enzyme in aliquid test sample, comprising the steps of:(a) contacting the testsample with a chromogenic enzyme substrate compound of the formula:##STR8## wherein W is O or S; R and R', which can be the same ordifferent, are H, alkyl, or aryl; and Y is an enzyme-cleavable groupthat is(i) capable of being cleaved from the nucleus of the enzymesubstrate compound by said enzyme, or (ii) capable of being modified bysaid enzyme to produce a secondary substrate compound in which Y iscapable of being cleaved from the nucleus of the modified enzymesubstrate compound by a secondary enzyme, and, in the latter case (ii),the secondary substrate compound is contacted with said secondaryenzyme; and (b) measuring and correlating the resulting color generatedby the cleaved nucleus of the enzyme substrate compound to the presenceof said enzyme in said liquid test sample.
 2. The method of claim 1wherein said enzyme-cleavable group is a radical of a compound Y--OHcomprising an enzyme-specific moiety selected from the group consistingof sugars and derivatives thereof, aliphatic and aromatic carboxylicacids, and inorganic acids.
 3. The method of claim 2 wherein W is O. 4.The method of claim 3 wherein R and R', which can be the same ordifferent, are H, lower alkyl, or phenyl.
 5. The method of claim 4wherein R and R' are not both H or phenyl.
 6. The method of claim 3wherein one of R and R' is H and the other is lower alkyl.
 7. The methodof claim 3 wherein one of R and R' is H and the other is methyl.
 8. Themethod of claim 3 wherein R and R' are both methyl.
 9. The method ofclaim 1 wherein said enzyme is a glycosidase and Y is a radical of asugar or derivative thereof selected from the group consisting ofα-D-galactose, β-D-galactose, α-D-glucose, α-D-glucose, α-D-mannose,N-acetylglucosamine and N-acetylneuraminic acid.
 10. The method of claim1 wherein said enzyme is α-amylase and Y is a radical of anoligosaccharide chain of from between about 2 to about 20 monosaccharideunits which is capable of being modified by α-amylase to produce asecondary glycoside substrate compound in which the resulting glycosidegroup is cleavable from the nucleus of the substrate compound by asecondary glycosidase enzyme, and wherein the secondary glycosidesubstrate compound is contacted with said secondary glycosidase enzyme.11. The method of claim 10 wherein said oligosaccharide chain ismaltoheptose.
 12. The method of claim 10 wherein said resultingglycoside group is a glucoside and said secondary glycosidase enzyme isa glucosidase.
 13. The method of claim 1 wherein said enzyme isβ-D-galactosidase and Y is a β-D-galactose radical.
 14. The method ofclaim 1 wherein said enzyme is β-D-glucosidase and Y is a β-D-glucoseradical.
 15. The method of claim 1 wherein said enzyme is a non-specificesterase and Y is a radical of an aliphatic or aromatic carboxylic acid.16. The method of claim 1 wherein said enzyme is a proteolytic enzymepresent in leukocytes and Y is a radical of a carboxylic acid comprisingan N-protected amino acid or a peptide of from between about 2 to about5 amino acid units.
 17. The method of claim 16 wherein Y is anN-tosyl-L-alanine radical.
 18. The method of claim 1 wherein said enzymeis alkaline phosphatase and Y is a phosphoric acid radical.
 19. Themethod of claim 1 wherein said enzyme is sulfatase and Y is a sulfuricacid radical.